Adeno-associated virus (AAV) is a single-stranded DNA parvovirus with a helper-dependent life cycle that replicates in the presence of helper viruses such as adenovirus or herpes virus (Berns et al., 1996; Carter et al., 2000). The type 2 AAV (AAV-2) DNA genome is 4681 nucleotides long and includes an identical 5′ and 3′ copy of a 145 nucleotide long inverted terminal repeat (ITR) and a unique sequence of 4391 nucleotides that contains two main open reading frames for the rep and cap genes (Srivastava et al., 1983; Smuda et al., 1991). This unique region contains three transcription promoters p5, p19, and p40 that are used to express the rep and cap genes. The ITRs contain palindromic sequences capable of forming a secondary stem loop structure and are required in cis to provide functional origins of replication (ori). In addition, AAV ITRS also provide sequences necessary for encapsidation, integration, and rescue of the rAAV from either host cell chromosomes or recombinant plasmids. AAV ITR sequences function in two distinct aspects. First, as described above, ITR sequences are required for AAV production, assembly and packaging. Secondly, AAV ITRs function in the persistence and expression of a linked transgene in a host cell.
In a productive AAV infection in the presence of a helper such as adenovirus, the infecting parental AAV single strand genome is converted to a parental duplex replicating form (RF) by a self-priming mechanism as a result of the ability the ability of the ITR to form a hairpin structure (Muzyczka, 1992; Carter et al., 1989). The parental RF molecule is then amplified to form a large pool of progeny RF molecules in a process that requires both helper functions and the AAV rep gene products, Rep78 and Rep68. AAV RF genomes are a mixture of head-to-head or tail-to-tail multimers or concatamers, and are precursors to progeny single strand (SS) DNA genomes that are packaged into pre-formed empty AAV capsids. Rep52 and Rep40 interact with the pre-formed capsid to provide a DNA helicase function for DNA packaging (Myers et al., 1980; King et al., 2001).
The kinetics of AAV replication and assembly has been investigated. In human HeLa or 293 cells simultaneously infected with AAV and adenovirus, there are three phases of the growth cycle. In the first 8 to 10 hours, the cell becomes permissive for AAV replication as a result of expression of a subset of adenovirus genes including E1, E2A, E4 and the VA RNA. During this period, the infecting AAV genome is converted to the initial parental duplex RF DNA by self-priming from the terminal base-paired 3′ hydroxyl group provided by the ITR, which can form a self-paired hairpin. This initial generation of a duplex genome also provides a template for transcription and expression of AAV proteins. In a second phase, from about 10 to 20 hours after infection, the bulk of the AAV rep and cap proteins are synthesized, and there is a large amplification of monomeric and concatameric duplex AAV RF genomes to a constant level. During the third phase of AAV growth, between 16 to 30 hours, single strand progeny molecules are synthesized by a strand-displacement replication mechanism and packaged into pre-formed capsids followed by accumulation of mature, infectious AAV particles (Redemann et al., 1998; Carter et al., 1989). Rep68 and Rep78 bind to the ITR and are site-specific, strand-specific endonucleases that cleave the hairpin in an RF molecule at the site that is the 5′ terminus of the mature strand. In addition, these proteins contain an ATP-binding site which is important for enzymatic activity but not for binding to the ITR. Further, Rep78 and 68 have both DNA and RNA helicase activity. Rep proteins also regulate transcription (Muzyczka; 1992; Carter et al., 1992; Flotte et al., 1995; Hallek et al., 1998; Flotte et al., 1996). Rep78 is a negative auto-regulator of the p5 promoter, i.e., of its own synthesis, but is an activator of the p40 promoter to enhance capsid protein production. Rep52 and Rep40 do not bind to the ITR but provide a helicase in assembly of mature particles. Also, the smaller rep proteins are anti-repressors and block the negative auto-regulation of p5 by Rep78. rAAV vectors which contain only the 5′ and 3′ ITR sequences flanked by the transgene of interest are replicated analogous to wild-type AAV except rep and cap functions are provided to the permissive cell in trans.
In the absence of selective pressure, recombinant AAV (rAAV) vectors generally persist as episomal genomes. A number of studies have now shown that rAAV vectors can persist for extended periods of time when administered in vivo and that the predominant form of the persisting vector genomes appears to be multimeric structures which are head-to-tail concatamers that are circular. How these head-to-tail multimers are formed is unknown but it cannot involve the normal AAV replication process because that requires rep protein and gives only head-to-head or tail-to-tail concatamers. Whether the circular concatemers are integration intermediates, as has been suggested for AAV integration, is also unknown. However, available evidence indicates that the majority of these head-to-tail concatamers are episomal and that integrated copies of vector in organs such as liver or muscle are very rare (Flotte et al., 1993; Afione et al., 1996; Kaplitt et al., 1994; Xiao et al., 1996; Kessler et al., 1996; Koerberl et al., 1997; Ponnazhagan et al., 1997; Zadori et al., 2001; Fisher et al., 1997; Herzog et al., 1997; Snyder et al., 1997; Miao et al., 1998; Duan et al., 1998; Duan et al., 1999; Vincent-Lacaze et al., 1999; McKeon et al., 1996; Nakai et al., 2001; Schnepp et al., 2003.)
rAAV-based vector systems have attracted great attention for human gene therapy for more than 10 years. Although rAAV vectors have now been generated from eight different serotypes (Chao et al., 2000; Grimm et al., 2003), rAAV-2 has been most extensively studied as a gene transfer vector, and has been used in clinical trials for cystic fibrosis (Flotte et al., 1996; Wagner et al., 1999; Wagner et al., 1998) and hemophilia B (Hagstrom et al., 2000; Kay et al., 2000). Over the last decade, knowledge of the molecular virology of AAV has expanded significantly. Insights into various aspects of receptor interactions, intercellular trafficking, and genome conversion have led to improvements in the design and performance of rAAV vector systems. For example, studies evaluating the molecular mechanisms of rAAV-2 genome conversion have uncovered unique mechanisms of latent viral genome persistence that involve linear and/or circular concatamerization (Duan et al., 1998; Yang et al., 1999). Studies evaluating the molecular mechanisms of rAAV genome concatamerization have demonstrated that this process occurs through intermolecular joining of two independent viral genomes as opposed to self-priming replication (Yang et al., 1999). This important discovery has lead to development of dual vector heterodimerization approaches to deliver transgene cassettes that exceed the 4.7 kb packaging limitation of rAAV vectors. This innovative approach utilizes two independent vectors to deliver separately encoded transgene exons to the same cell, which vectors are capable of reconstituting a transgene product by trans-splicing across heterodimer genomes formed by intermolecular recombination (Yan et al., 2002). Such an approach has effectively doubled the capacity of a single AAV vector and has been successfully tested with reporter genes in muscle (Duan et al., 2000; Sun et al., 2000; Yan et al., 2000), liver (Nakai et al., 2003), eyes (Reich et al., 2003), and lung. These studies have paved the way for applications of dual trans-splicing vectors for diseases such as cystic fibrosis, Duchenne muscular dystrophy, and hemophilia A, all of which require the delivery of large cDNA expression cassettes which approach or exceed the packaging capacity of a single rAAV vector.
Despite the promising prospects of dual rAAV vector approaches, the level of gene expression from the current system remains substantially lower than delivery of a transgene encoded within a single vector. Several factors currently limit the efficiency of dual vector heterodimerization approaches. First, the multiplicity of infection must be high enough such that both delivered vectors infect the same cell. Second, in the context of dual vector trans-splicing approaches, the generation of a functional transgene is directionally dependent on the joining of two viral genomes in the correct orientation. For dual vectors containing two minigene exons in the same orientation, only heterodimers formed by a tail to head (T-H) recombination event are capable of yielding an active reconstituted transgene product via trans-splicing. While approaches aimed at increasing the ratio of T-H oriented heterodimers and larger concatamers could conceivably improve the efficiency of dual vector trans-splicing delivery of transgene products, the mechanisms responsible for intermolecular recombination of rAAV genomes remain poorly understood. Previous studies in muscle have suggested that monomer circular viral genome intermediates with double-D structure may be precursors to the formation of episomally stable concatamers (Duan et al., 1998; Yan et al., 2000). In these studies, intermolecular recombination appeared to occur between monomer circular genomes in a time-dependent manner leading to high molecular weight circular concatamers. In contrast, studies in liver with rAAV and linear AAV-like genomes have suggested that concatamerization may be independent of the ITR and primarily involve linear intermolecular end-end ligation (Nakai et al., 2003; Nakai et al., 2000). A clear delineation of these two mechanisms as well as potential tissue-specific differences in the molecular structure of concatamers may be important to improve the efficiency of rAAV dual vector approaches.
What is needed is a method to increase the efficiency of rAAV vectors in general as well as rAAV dual vector technologies.